CN117402160A - Wnt pathway inhibitor compound - Google Patents

Wnt pathway inhibitor compound Download PDF

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Publication number
CN117402160A
CN117402160A CN202311342585.0A CN202311342585A CN117402160A CN 117402160 A CN117402160 A CN 117402160A CN 202311342585 A CN202311342585 A CN 202311342585A CN 117402160 A CN117402160 A CN 117402160A
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compound
reaction
dissolved
mmol
white solid
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陈宇锋
武朋
吕萌
刘灿丰
陈凯旋
杨寒
程万里
王友平
路萍萍
何南海
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Hangzhou Arnold Biomedical Technology Co ltd
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    • A61K31/395—Heterocyclic compounds having nitrogen as a ring hetero atom, e.g. guanethidine or rifamycins
    • A61K31/495—Heterocyclic compounds having nitrogen as a ring hetero atom, e.g. guanethidine or rifamycins having six-membered rings with two or more nitrogen atoms as the only ring heteroatoms, e.g. piperazine or tetrazines
    • A61K31/4985—Pyrazines or piperazines ortho- or peri-condensed with heterocyclic ring systems
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    • A61K31/395—Heterocyclic compounds having nitrogen as a ring hetero atom, e.g. guanethidine or rifamycins
    • A61K31/495—Heterocyclic compounds having nitrogen as a ring hetero atom, e.g. guanethidine or rifamycins having six-membered rings with two or more nitrogen atoms as the only ring heteroatoms, e.g. piperazine or tetrazines
    • A61K31/50—Pyridazines; Hydrogenated pyridazines
    • A61K31/5025—Pyridazines; Hydrogenated pyridazines ortho- or peri-condensed with heterocyclic ring systems
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
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    • A61K31/00—Medicinal preparations containing organic active ingredients
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    • A61K31/505—Pyrimidines; Hydrogenated pyrimidines, e.g. trimethoprim
    • A61K31/517—Pyrimidines; Hydrogenated pyrimidines, e.g. trimethoprim ortho- or peri-condensed with carbocyclic ring systems, e.g. quinazoline, perimidine
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    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
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    • A61K31/55—Heterocyclic compounds having nitrogen as a ring hetero atom, e.g. guanethidine or rifamycins having seven-membered rings, e.g. azelastine, pentylenetetrazole
    • A—HUMAN NECESSITIES
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    • A—HUMAN NECESSITIES
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    • C07D471/00—Heterocyclic compounds containing nitrogen atoms as the only ring hetero atoms in the condensed system, at least one ring being a six-membered ring with one nitrogen atom, not provided for by groups C07D451/00 - C07D463/00
    • C07D471/12—Heterocyclic compounds containing nitrogen atoms as the only ring hetero atoms in the condensed system, at least one ring being a six-membered ring with one nitrogen atom, not provided for by groups C07D451/00 - C07D463/00 in which the condensed system contains three hetero rings
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    • C07D487/12—Heterocyclic compounds containing nitrogen atoms as the only ring hetero atoms in the condensed system, not provided for by groups C07D451/00 - C07D477/00 in which the condensed system contains three hetero rings
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Abstract

The invention relates to a Wnt channel inhibitor compound shown in a formula 25, a synthesis method thereof, a pharmaceutical composition containing the compound, and application of the compound shown in the formula 25 in preventing and/or treating cancers, tumors, inflammatory diseases, autoimmune diseases or immune-mediated diseases.

Description

Wnt pathway inhibitor compound
The present application is a divisional application of the Chinese patent application with the application number of 202280013024. X. The application date of the parent application is 2022, 7 and 26 (PCT International application date), the Chinese national application number is 202280013024. X (PCT International application number is PCT/CN 2022/107727), and the invention is named as a Wnt channel inhibitor compound.
The parent application and the present divisional application claim priority to chinese patent application 202110847130.9 entitled "a Wnt pathway inhibitor compound" filed on the title of the chinese national intellectual property agency at day 7, 2021, 26, the contents of which are incorporated herein by reference in their entirety.
Technical Field
The invention relates to a heterocyclic compound, in particular to a high-activity Wnt channel inhibitor, a preparation method and application thereof.
Background
The Wnt/beta-catenin signal transduction pathway is a pathway conserved in biological evolution. In normal somatic cells, β -catenin acts only as a cytoskeletal protein complex with E-cadherein at the cell membrane to maintain cell adhesion of the isotype and prevent cell migration. When the Wnt signal channel is not activated, the beta-catenin in cytoplasm is phosphorylated and forms a beta-catenin degradation complex with APC, axin, GSK3 beta and the like, so that the ubiquitin system is started to degrade the beta-catenin through a proteasome way, and the beta-catenin in cytoplasm is maintained at a lower level. When the cell is stimulated by Wnt signals, the Wnt protein is combined with a specific receptor Frizzled protein on a cell membrane, the activated Frizzled receptor recruits an intracellular disheveled protein, the degradation activity of a beta-catenin degradation complex formed by GSK3 beta and other proteins is inhibited, and the beta-catenin protein in a free state in cytoplasm is stabilized. The stably accumulated beta-catenin in cytoplasm enters the nucleus and then binds with LEF/TCF transcription factor family to start the transcription of downstream target genes (such as c-myc, c-jun, cyclin D1, etc.). Overactivation of the Wnt/β -catenin signaling pathway is closely associated with the occurrence of a variety of cancers (including colon, stomach, breast, etc.). For example, abnormal activation of Wnt canonical signaling pathway and nuclear accumulation of β -catenin protein are widely present in colorectal cancer, and proliferation of cancers such as colon cancer can be inhibited by inhibiting Wnt signaling pathway activity. Mutations of APC exist in more than 85% of colorectal cancers, and the mutated APC blocks the phosphorylation degradation of beta-catenin to induce the occurrence of colorectal cancers. In addition, the mutation of Axin and the mutation of beta-catenin can cause the intracellular aggregation of beta-catenin and activate Wnt/beta-catenin channels.
While inhibition of the Wnt signaling pathway is known to be effective in preventing and/or treating cancer, tumors, inflammatory diseases, autoimmune diseases, and immune-mediated diseases, there is currently no satisfactory effective Wnt pathway inhibitor compound in the art. Thus, there is a need in the art to investigate effective Wnt pathway inhibitor compounds.
Disclosure of Invention
In one aspect, the invention provides a compound that inhibits Wnt pathway activity having the structure of formula 25, or a pharmaceutically acceptable salt, isotopic derivative, stereoisomer thereof:
in another aspect, the present invention provides a method of preparing a compound having the structure of formula 25, comprising the steps of:
the first step: compound 1b, compound 14d and cesium carbonate were dissolved in N, N-dimethylformamide, stirred at 80 ℃ until the reaction was completed, the reaction solution was diluted with ethyl acetate, washed with water and saturated brine in this order, the organic phase was dried over anhydrous sodium sulfate, the reaction solution was concentrated, and the residue was purified by silica gel column chromatography (dichloromethane/methanol=20/1) to give white solid 25b; and a second step of: compound 25b is dissolved in dichloromethane, 1, 4-dioxane solution of hydrochloric acid is added dropwise, the mixture is stirred at room temperature until the reaction is finished, and the reaction solution is concentrated to obtain white solid 25c; and a third step of: compound 1i, compound 25c and p-toluenesulfonic acid monohydrate were dissolved in n-butanol and reacted at 160 ℃ under microwave to completion, and the reaction solution was purified by reverse phase preparative HPLC to give white solid 25.
Further, the invention also provides a pharmaceutical composition comprising the compound or a pharmaceutically acceptable salt, an isotope derivative or a stereoisomer thereof.
Further, the invention also provides application of the compound or pharmaceutically acceptable salt, isotopic derivative, stereoisomer or the pharmaceutical composition of the invention in preparing medicines for preventing and/or treating cancers, tumors, inflammatory diseases, autoimmune diseases or immune-mediated diseases. It is particularly noted that, in this context, when referring to "compounds" of the structure of formula 25, stereoisomers, diastereomers, enantiomers, racemic mixtures, and isotopic derivatives thereof are also generally contemplated.
Accordingly, the present invention provides a method for preventing and/or treating cancer, tumor, inflammatory disease, autoimmune disease or immune-mediated disease, comprising administering to a subject a compound of the present invention or a pharmaceutically acceptable salt, isotopic derivative, stereoisomer or a pharmaceutical composition of the present invention.
It is well known to those skilled in the art that salts, solvates, hydrates of a compound are alternative forms of the compound, which can all be converted to the compound under certain conditions, and therefore, particular attention is paid herein to the compound of formula 25, generally also including pharmaceutically acceptable salts thereof, and further including solvates and hydrates thereof.
Similarly, when a compound is referred to herein, prodrugs, metabolites, and nitrogen oxides thereof are also generally included.
Pharmaceutically acceptable salts according to the invention may be formed using, for example, the following mineral or organic acids: by "pharmaceutically acceptable salt" is meant a salt which is, within the scope of sound medical judgment, suitable for use in contact with the tissues of humans and lower animals without undue toxicity, irritation, allergic response and the like commensurate with a reasonable benefit/risk ratio. The salts can be prepared in situ during the final isolation and purification of the compounds of the invention, or by reacting the free base or free acid with a suitable reagent alone, as outlined below. For example, the free base function may be reacted with a suitable acid. Examples of pharmaceutically acceptable inorganic acid addition salts are salts of amino groups with inorganic acids (e.g., hydrochloric, hydrobromic, phosphoric, sulfuric and perchloric) or organic acids (e.g., acetic, oxalic, maleic, tartaric, citric, succinic or malonic) or by using other methods in the art such as ion exchange. Other pharmaceutically acceptable salts include non-toxic ammonium salts, quaternary ammonium salts, and amine cations formed with counterions, such as halides, hydroxides, carboxylates, sulfates, phosphates, nitrates, lower alkyl sulfonates, and aryl sulfonates, as appropriate.
The pharmaceutically acceptable salts of the invention may be prepared by conventional methods, for example by dissolving the compounds of the invention in a water miscible organic solvent (e.g. acetone, methanol, ethanol and acetonitrile), adding thereto an excess of an organic or inorganic acid aqueous solution to precipitate the salt from the resulting mixture, removing the solvent and the remaining free acid therefrom, and then isolating the precipitated salt.
The precursors or metabolites of the invention may be precursors or metabolites well known in the art, as long as the precursors or metabolites are converted into compounds by in vivo metabolism. For example, "prodrugs" refer to those prodrugs of the compounds of the present invention which are, within the scope of sound medical judgment, suitable for use in contact with the tissues of humans and lower animals without undue toxicity, irritation, allergic response and the like commensurate with a reasonable benefit/risk ratio, and are effective for their intended use. The term "prodrug" refers to a compound that is rapidly transformed in vivo to produce the parent compound of the formula described above, for example by metabolism in vivo, or N-demethylation of a compound of the invention.
"solvate" as used herein means a physical association of a compound of the invention with one or more solvent molecules (whether organic or inorganic). The physical association includes hydrogen bonding. In some cases, for example when one or more solvent molecules are incorporated into the crystal lattice of a crystalline solid, the solvate will be able to be isolated. The solvent molecules in the solvate may be present in a regular arrangement and/or in a disordered arrangement. The solvate may comprise a stoichiometric or non-stoichiometric solvent molecule. "solvate" encompasses both solution phases and separable solvates. Exemplary solvates include, but are not limited to, hydrates, ethanolates, methanolates, and isopropanolamides. Solvation methods are well known in the art.
The term "stereoisomers" as used herein is divided into conformational isomerism and configurational isomerism, which may be also divided into cis-trans isomerism and optical isomerism (i.e. optical isomerism), and conformational isomerism refers to a stereoisomerism phenomenon that an organic molecule with a certain configuration makes each atom or group of molecules generate different arrangement modes in space due to rotation or twisting of carbon and carbon single bonds, and commonly includes structures of alkane and cycloalkane compounds, such as chair-type conformations and boat-type conformations, which occur in cyclohexane structures. "stereoisomers" means that when a compound of the invention contains one or more asymmetric centers, it is useful as racemate and racemic mixtures, single enantiomers, diastereomeric mixtures and individual diastereomers. The compounds of the invention have asymmetric centers, each of which produces two optical isomers, and the scope of the invention includes all possible optical isomers and diastereomeric mixtures and pure or partially pure compounds. The compounds described herein may exist in tautomeric forms having different points of attachment of hydrogen through displacement of one or more double bonds. For example, the ketone and its enol form are keto-enol tautomers. Each tautomer and mixtures thereof are included in the compounds of the present invention. All enantiomers, diastereomers, racemates, meso, cis-trans isomers, tautomers, geometric isomers, epimers, mixtures thereof and the like of the compounds of formula 25 are included within the scope of the present invention.
The term "isotopically-labeled" as used herein refers to molecules wherein the compound is isotopically labeled. Isotopes commonly used as isotopic labels are: the hydrogen isotope is selected from the group consisting of, 2 h and 3 h is formed; carbon isotopes: 11 C, 13 c and C 14 C, performing operation; chlorine isotopes: 35 cl and Cl 37 Cl; fluorine isotopes: 18 f, performing the process; iodine isotopes: 123 i and 125 i, a step of I; nitrogen isotopes: 13 n and 15 n; oxygen isotopes: 15 O, 17 o and 18 isotopes of O and sulfur 35 S, S. These isotopically-labeled compounds can be used to study the distribution of a pharmaceutical molecule in a tissue. In particular deuterium 3 H and carbon 13 C, because they are easily labeled and conveniently detected, the application is wider. Certain heavy isotopes, such as heavy hydrogen @, for example 2 H) Substitution energy increase of (C)The stability of metabolism is strong, the half life period is prolonged, and the aim of reducing dosage is achieved, so that the curative effect is improved. Isotopically-labeled compounds generally begin with a starting material that has been labeled, and are synthesized using known synthetic techniques like synthesizing non-isotopically-labeled compounds.
The invention also provides the use of the compounds of the invention in the manufacture of a medicament for the prophylaxis and/or treatment of cancer, tumour, inflammatory disease, autoimmune disease or immune mediated disease.
Furthermore, the present invention provides a pharmaceutical composition for preventing and/or treating cancer, tumor, inflammatory disease, autoimmune disease, neurodegenerative disease, attention-related disease or immune-mediated disease, comprising the compound of the present invention as an active ingredient. The pharmaceutical composition may optionally comprise a pharmaceutically acceptable carrier.
Furthermore, the present invention provides a method for preventing and/or treating cancer, tumor, inflammatory disease, autoimmune disease, neurodegenerative disease, attention-related disease or immune-mediated disease comprising administering to a mammal in need thereof a compound of the present invention.
The compounds of the present invention or pharmaceutically acceptable salts thereof may provide enhanced anticancer effects when administered in combination with additional anticancer agents or immune checkpoint inhibitors for the treatment of cancer or tumors.
The compounds of the present invention or pharmaceutically acceptable salts thereof may provide enhanced therapeutic effects when administered in combination with additional therapeutic agents for the treatment of inflammatory, autoimmune and immune-mediated diseases.
The compounds of the present invention or pharmaceutically acceptable salts thereof may be administered orally or parenterally as an active ingredient. The dosage of the active ingredient may be adjusted according to a number of relevant factors, such as the condition of the subject to be treated, the type and severity of the disease, the rate of administration and the opinion of the physician. In some cases, amounts less than the above dosages may be suitable. An amount greater than the above dosage may be used if it does not cause deleterious side effects and may be administered in divided doses per day.
In addition, the present invention provides a method for preventing and/or treating a tumor, cancer, viral infection, organ transplant rejection, neurodegenerative disease, attention-related disease or autoimmune disease, comprising administering to a mammal in need thereof a compound of the present invention or a pharmaceutical composition of the present invention.
The pharmaceutical compositions of the present invention may be formulated according to any of the conventional methods into dosage forms for oral administration or parenteral administration (including intramuscular, intravenous and subcutaneous routes, intratumoral injection), such as tablets, granules, powders, capsules, syrups, emulsions, microemulsions, solutions or suspensions.
The pharmaceutical compositions of the invention for oral administration can be prepared by mixing the active ingredient with, for example, the following carriers: cellulose, calcium silicate, corn starch, lactose, sucrose, dextrose, calcium phosphate, stearic acid, magnesium stearate, calcium stearate, gelatin, talc, surfactants, suspending agents, emulsifying agents and diluents.
Examples of carriers employed in the pharmaceutical compositions for injectable administration of the present invention may be water, salt solutions, dextrose-like solutions (glucose), alcohols, glycols, ethers (e.g., polyethylene glycol 400), oils, fatty acids, fatty acid esters, glycerides, surfactants, suspending agents and emulsifiers.
Other features of the present invention will become apparent in the course of describing exemplary embodiments of the invention, which are presented to illustrate the invention and are not intended to be limiting thereof, the following examples being prepared, isolated and characterized using the methods disclosed herein.
The compounds of the present invention may be prepared in a variety of ways known to those skilled in the art of organic synthesis, and may be synthesized using the methods described below as well as synthetic methods known in the art of organic synthetic chemistry or by variations thereof as will be appreciated by those skilled in the art. Preferred methods include, but are not limited to, those described below. The reaction is carried out in a solvent or solvent mixture suitable for the kit materials used and for the transformation to be effected. Those skilled in the art of organic synthesis will understand that the functionalities present on the molecule are consistent with the proposed transformations. This sometimes requires judgment to change the order or starting materials of the synthesis steps to obtain the desired compounds of the invention.
Detailed Description
Terminology
The terms used in the present application, including the specification and claims, are defined as follows, unless otherwise indicated. Conventional methods of mass spectrometry, nuclear magnetism, HPLC, protein chemistry, biochemistry, recombinant DNA techniques and pharmacology are used, if not otherwise indicated. In this application, the use of "or" and "means" and/or "unless otherwise indicated.
In the description and claims, a given formula or name shall encompass all stereoisomers and optical isomers thereof, as well as racemates in which the above isomers exist. Unless otherwise indicated, all chiral (enantiomers and diastereomers) and racemic forms are within the scope of the present invention. Various geometric isomers of c=c double bonds, c=n double bonds, ring systems, etc. may also be present in the compounds, and all such stable isomers are contemplated within the present invention. The present invention describes cis-and trans- (or E-and Z-) geometric isomers of the compounds of the present invention, and which may be separated into mixtures of isomers or separate isomeric forms. The compounds of the invention may be isolated in optically active or racemic forms. All processes for preparing the compounds of the invention and intermediates prepared therein are considered part of the present invention. When preparing the enantiomeric or diastereomeric products, they can be separated by conventional methods, for example by chromatography or fractional crystallization. Depending on the process conditions, the end products of the invention are obtained in free (neutral) or salt form. Both the free form and the salt of these end products are within the scope of the invention. If desired, one form of the compound may be converted to another form. The free base or acid may be converted to a salt; the salt may be converted to the free compound or another salt; mixtures of the isomeric compounds of the invention may be separated into the individual isomers. The compounds of the invention, free forms and salts thereof, may exist in various tautomeric forms in which hydrogen atoms are transposed to other parts of the molecule and thereby the chemical bonds between the atoms of the molecule are rearranged. It is to be understood that all tautomeric forms that may exist are included within the invention.
The term "patient" as used herein refers to an organism treated by the methods of the present invention. Such organisms preferably include, but are not limited to, mammals (e.g., murine, simian, monkey, horse, bovine, porcine, canine, feline, etc.) and most preferably refer to humans.
The term "effective amount" as used herein means the amount of a drug or pharmaceutical agent (i.e., a compound of the present invention) that will elicit the biological or medical response of a tissue, system, animal or human that is being sought, for instance, by a researcher or clinician. Furthermore, the term "therapeutically effective amount" means an amount of: such amounts result in improved treatment, cure, prevention, or alleviation of a disease, disorder, or side effect, or a reduction in the rate of progression of a disease or disorder, as compared to a corresponding subject not receiving such amounts. An effective amount may be administered in one or more administrations, or dosages and is not intended to be limited to a particular formulation or route of administration. The term also includes within its scope an effective amount to enhance normal physiological function.
The term "treatment" as used herein includes any effect that results in an improvement in a condition, disease, disorder, etc., such as a reduction, decrease, modulation, improvement or elimination, or improvement of symptoms thereof.
The term "pharmaceutically acceptable" is used herein to refer to those compounds, materials, compositions, and/or dosage forms which are: it is suitable for use in contact with human and animal tissue without undue toxicity, irritation, allergic response, and/or other problems or complications commensurate with a reasonable benefit/risk ratio, within the scope of sound medical judgment.
The phrase "pharmaceutically acceptable carrier" as used herein means a pharmaceutical substance, composition or vehicle, such as a liquid or solid filler, diluent, excipient, manufacturing aid (e.g., lubricant, talc, magnesium stearate, calcium or zinc stearate, or stearic acid), or solvent encapsulating material, which involves carrying or transporting the subject compound from one organ or body part to another organ or body part. Each carrier must be "acceptable" in the sense of being compatible with the other ingredients of the formulation and not deleterious to the patient.
The term "pharmaceutical composition" means a composition comprising a compound of the invention and at least one other pharmaceutically acceptable carrier. "pharmaceutically acceptable carrier" refers to a medium commonly accepted in the art for delivery of biologically active agents to animals, particularly mammals, and includes (i.e., adjuvants, excipients or vehicles such as diluents, preservatives, fillers, flow control agents, disintegrants, wetting agents, emulsifying agents, suspending agents, sweetening agents, flavoring agents, antibacterial agents, antifungal agents, lubricants, and dispersing agents, depending upon the mode of administration and the nature of the dosage form.
The term "acceptable" as used herein, means that a prescription component or active ingredient does not unduly adversely affect the health of the general therapeutic objective.
The term "cancer", as used herein, refers to an abnormal growth of cells that is not controllable and is capable of metastasis (transmission) under certain conditions. Cancers of this type include, but are not limited to, solid tumors (e.g., bladder, intestine, brain, chest, uterus, heart, kidney, lung, lymphoid tissue (lymphoma), ovary, pancreas, or other endocrine organ (e.g., thyroid), prostate, skin (melanoma), or hematological tumors (e.g., non-leukemia).
The term "co-administration" or similar terms, as used herein, refers to administration of several selected therapeutic agents to a patient, administered at the same or different times, in the same or different modes of administration.
The term "enhance" or "potentiating," as used herein, means that the intended result can be increased or prolonged in either efficacy or duration. Thus, in enhancing the therapeutic effect of a drug, the term "capable of enhancing" refers to the ability of the drug to increase or prolong the efficacy or duration of the drug in the system. As used herein, "potentiating value" means that the ability of another therapeutic agent to be maximally enhanced in an ideal system.
The term "immunological disorder" refers to a disease or condition that produces an adverse or detrimental response to an endogenous or exogenous antigen. As a result, the cells are often dysfunctional, or thus destroyed and dysfunctional, or destroy organs or tissues that may develop immune symptoms.
The term "kit" is synonymous with "product package".
The term "subject" or "patient" includes mammals and non-mammals. Mammals include, but are not limited to, mammals: humans, non-human primates such as gorillas, apes, and monkeys; agricultural animals such as cattle, horses, goats, sheep, pigs; domestic animals such as rabbits and dogs; laboratory animals include rodents such as rats, mice, guinea pigs, and the like. Non-mammalian animals include, but are not limited to, birds, fish, and the like. In a preferred aspect, the mammal selected is a human.
The terms "treat," "course of treatment," or "therapy" as used herein include alleviation, inhibition, or amelioration of symptoms or conditions of a disease; inhibit the occurrence of complications; improving or preventing underlying metabolic syndrome; inhibiting the occurrence of a disease or condition, such as controlling the progression of a disease or condition; alleviating a disease or symptom; causing the disease or symptom to subside; alleviating complications caused by diseases or symptoms, or preventing and/or treating signs caused by diseases or symptoms.
As used herein, a compound or pharmaceutical composition, upon administration, may result in an improvement in a disease, symptom, or condition, particularly an improvement in severity, delay of onset, slow progression, or decrease in duration. Whether stationary or temporary, continuous or intermittent, may be due to or associated with administration.
Route of administration
Suitable routes of administration include, but are not limited to, oral, intravenous, rectal, aerosol, parenteral, ocular, pulmonary, transdermal, vaginal, auditory canal, nasal, and topical. Further, by way of example only, parenteral administration includes intramuscular, subcutaneous, intravenous, intramedullary, ventricular, intraperitoneal, intralymphatic, and intranasal.
In one aspect, the administration of the compounds described herein is topical rather than systemic. In particular embodiments, the depot is administered by implantation (e.g., subcutaneously or intramuscularly) or by intramuscular injection. Furthermore, in another specific embodiment, the drug is administered by a targeted drug delivery system. For example, liposomes encapsulated by organ-specific antibodies. In this particular embodiment, the liposomes are selectively targeted to a specific organ and absorbed.
The scope of the present invention includes pharmaceutical compositions (alone or in combination with a pharmaceutical carrier) comprising a therapeutically effective amount of at least one compound of the present invention as an active ingredient. Optionally, the compounds of the present invention may be used alone, in combination with other compounds of the present invention, or in combination with one or more other therapeutic agents (e.g., anticancer agents or other pharmaceutically active substances).
Regardless of the route of administration selected, the compounds of the invention (which may be used in a suitable hydrated form) and/or the pharmaceutical compositions of the invention are formulated into pharmaceutical dosage forms by conventional methods known to those skilled in the art.
The actual dosage level of the active ingredient in the pharmaceutical compositions of the present invention may be varied to achieve amounts of the active ingredient that are effective to achieve the desired therapeutic response, composition and mode of administration for a particular patient, but which are non-toxic to the patient.
The selected dosage level will depend on a variety of factors including the activity of the particular compound of the invention or an ester, salt or amide thereof employed; a route of administration; administration time; the rate of excretion of the particular compound being used; the rate and extent of absorption; duration of treatment; other drugs, compounds and/or substances used in combination with the particular compound used; the age, sex, weight, condition, general health and previous medical history of the patient being treated.
Although the compounds of the present invention may be administered alone, it is preferable to administer the compounds in the form of a pharmaceutical formulation (composition).
Kit/product package
All of the features described in this specification (including any accompanying claims, abstract) and/or all of the steps of any method or process so described, may be present in any combination, unless certain features or steps are mutually exclusive in the same combination.
The above-mentioned features of the invention, or of the embodiments, may be combined in any desired manner. All of the features disclosed in this specification may be combined with any combination of the features disclosed in this specification, and the various features disclosed in this specification may be substituted for any alternative feature serving the same, equivalent or similar purpose. The disclosed features are thus merely representative of general examples of equivalent or similar features, unless specified otherwise.
The invention will be further illustrated with reference to specific examples. It is to be understood that these examples are illustrative of the present invention and are not intended to limit the scope of the present invention. The experimental procedures, which do not address the specific conditions in the examples below, are generally carried out under conventional conditions or under conditions recommended by the manufacturer. All percentages, ratios, proportions, or parts are by weight unless otherwise indicated.
The units in weight volume percent are well known to those skilled in the art and refer, for example, to the weight of solute (g) in 100 milliliters of solution. Unless defined otherwise, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art. In addition, any methods and materials similar or equivalent to those described herein can be used in the methods of the present invention. The preferred methods and materials described herein are presented for illustrative purposes only.
Examples
Universal procedure
When the preparation route is not included, the raw materials and reagents used in the present invention are known products, and can be synthesized according to the methods known in the art, or can be obtained by purchasing commercial products. The commercial reagents used were all used without further purification.
Room temperature refers to 20-30 ℃.
The reaction examples are not particularly described, and the reactions are all carried out under nitrogen atmosphere. The nitrogen atmosphere is defined as the reaction flask being attached to a balloon of about 1L of nitrogen.
The hydrogenation reaction is usually vacuumized, filled with hydrogen and repeatedly operated for 3 times. The hydrogen atmosphere is defined as the reaction flask being connected to a balloon of hydrogen gas of about 1L.
Microwave reaction is usedInitiator + microwave reactor.
The structure of the compounds of the present invention is determined by Nuclear Magnetic Resonance (NMR) and Mass Spectrometry (MS). NMR shift (. Delta.) of 10 -6 Units of (ppm) are given. NMR was determined using (Bruker Assetnd TM 500) nuclear magnetic resonance apparatus, the measuring solvent is deuterated dimethyl sulfoxide (DMSO-d 6), deuterated chloroform (CDCl) 3 ) Deuterated methanol (CD) 3 OD), internal standard is Tetramethylsilane (TMS). The following abbreviations are used for multiplicity of NMR signals: s=singlet, brs=broad, d=doublet, t=triplet, m=multiplet. Coupling constants are listed as J values, measured in Hz.
LC-MS was determined using a Thermo liquid chromatography apparatus (UltiMate 3000+MSQ PLUS). HPLC was determined using a Thermo high pressure liquid chromatograph (UltiMate 3000). Reverse phase preparative chromatography a Thermo (UltiMate 3000) reverse phase preparative chromatograph was used. Quick column chromatography using Ai Jieer (FS-9200T) automatic column passing machine, silica gel pre-packed column using SantaiAnd (5) preassembling the column. The specification of the thin layer chromatography separation and purification product adopted by the smoke table yellow sea HSGF254 or Qingdao GF254 silica gel plate is 0.4 mm-0.5 mm.
Example 1
(S) -4, 5-dimethyl-2- ((trans-3- (3, 4, 5-trifluorophenoxy) cyclobutyl) amino) -4,5,9, 10-tetrahydro
-6h,8 h-pyrido [3,2, 1-des ] pteridin-6-one
Compound 1 was prepared by the following steps:
the first step: cis-3-BOC-aminocyclobutanol 1a (250 mg,1.34 mmol), methylsulfonic anhydride (463mg, 2.67 mmol) and N, N-diisopropylethylamine (517 mg,4.01 mmol) were dissolved in dichloromethane (2 mL) and stirred overnight at room temperature. TLC monitored the end of the reaction, the reaction was diluted with dichloromethane, washed successively with water and saturated brine, and the organic phase was dried over anhydrous sodium sulfate, filtered and concentrated to give yellow solid 1b (300 mg, yield 84%). 1 H NMR(500MHz,DMSO-d6)δ7.23(d,J＝8.3Hz,1H),4.69-4.64(m,1H),3.63-3.60(m,1H),3.13(s,3H),2.70-2.62(m,2H),2.16-2.09(m,2H),1.37(s,9H)。
And a second step of: compound 1b (300 mg,1.13 mmol), compound 1c (251 mg,1.70 mmol) and cesium carbonate (737 mg,2.26 mmol) were dissolved in N, N-dimethylformamide (2 mL) and stirred overnight at 80 ℃. LCMS monitored the end of the reaction, the reaction was diluted with ethyl acetate, washed sequentially with water and saturated brine, the organic phase was dried over anhydrous sodium sulfate, filtered and concentrated, and the residue was purified by silica gel column chromatography (dichloromethane/methanol=20/1) to give 1d (280 mg, 78% yield) as a white solid. ESI-MS (m/z): 318.6[ M+H ]] + 。
And a third step of: compound 1d (280 mg, 882. Mu. Mol) was dissolved in methylene chloride (2 mL), and 1, 4-dioxane solution (4M, 1.10 mL) of hydrochloric acid was added dropwise, followed by stirring overnight at room temperature. LCMS monitored the end of the reaction and the reaction concentrated to give 1e as a white solid (170 mg, 75% yield). ESI-MS (m/z): 218.4[ M+H ]] + 。
Fourth step: 2, 4-dichloropyrido [3,2-d ]]Pyrimidine 1f (1.7 g,8.50 mmol) and methyl (S) -2- (methylamino) propionate hydrochloride 1g (1.70 g,11.05 mmol) were dissolved in tetrahydrofuran (40 mL), triethylamine (2.58 g,25.50mmol,3.53 mL) was added, and stirred overnight at room temperature. LCMS monitored the end of the reaction, the reaction was concentrated and the residue was purified by column chromatography on silica gel to give a yellow oil for 1h (1.1 g, 46% yield). ESI-MS (m/z): 281.2[ M+H ] ] + 。
Fifth step:compound 1h (1.1 g,3.92 mmol) was dissolved in tetrahydrofuran (20 mL), aqueous hydrochloric acid (6N, 0.65 mL) and platinum dioxide (88 mg,0.39 mmol) were added, the reaction system was replaced with hydrogen gas with a hydrogen balloon, and stirred at room temperature under hydrogen balloon pressure for 48 hours, and LCMS monitored the reaction to completion. The reaction solution was diluted with methanol, filtered, and the filtrate was concentrated and purified by silica gel column chromatography to give 1i (900 mg, yield 90%) as a white solid. ESI-MS (m/z): 253.2[ M+H ]] + 。
Sixth step: compound 1i (50 mg, 197umol), compound 1e (65 mg,257 umol) and p-toluenesulfonic acid monohydrate (3.7 mg,19 umol) were dissolved in n-butanol (2 mL) and reacted at microwave 160℃for 2 hours. LCMS monitored the end of the reaction. The reaction solution was purified by reverse phase preparative HPLC to give 1 as a white solid (13 mg, yield 15%). ESI-MS (m/z): 434.3[ M+H ]] + ； 1 H NMR(500MHz,DMSO-d6)δ6.90-6.82(m,3H),4.86-4.81(m,1H),4.42-4.36(m,1H),4.12(q,J＝6.8Hz,1H),4.05-4.00(m,1H),3.30-3.24(m,1H),2.94(s,3H),2.55-2.52(m,2H),2.48-2.38(m,2H),2.36-2.28(m,2H),1.97-1.88(m,1H),1.85-1.76(m,1H),1.23(d,J＝6.8Hz,3H)。
Example 2
(S) -4, 5-dimethyl-2- ((trans-3- ((6- (trifluoromethyl) pyridin-3-yl) oxo) cyclobutyl) ammonia
Phenyl) -4,5,9, 10-tetrahydro-6H, 8H-pyrido [3,2, 1-des ] pteridin-6-one
Compound 2 was prepared by the following steps:
the first step: cis-3-BOC-aminocyclobutanol 1a (250 mg,1.34 mmol), methylsulfonic anhydride (463mg, 2.67 mmol) and N, N-diisopropylethylamine (517 mg,4.01 mmol) were dissolved in dichloromethane (2 mL) and stirred overnight at room temperature. TLC monitoring of the end of the reaction, dilution of the reaction solution with dichloromethane, washing with water and saturated brine in this order, drying the organic phase over anhydrous sodium sulfate Drying and concentration by filtration gave 1b (300 mg, yield 84%) as a yellow solid. 1 H NMR(500MHz,DMSO-d6)δ7.23(d,J＝8.3Hz,1H),4.69-4.64(m,1H),3.63-3.60(m,1H),3.13(s,3H),2.70-2.62(m,2H),2.16-2.09(m,2H),1.37(s,9H)。
And a second step of: compound 1b (500 mg,1.88 mmol), compound 2a (463mg, 2.83 mmol) and cesium carbonate (1.23 g,3.77 mmol) were dissolved in N, N-dimethylformamide (2 mL) and stirred overnight at 80 ℃. LCMS monitored the end of the reaction, the reaction was diluted with ethyl acetate, washed sequentially with water and saturated brine, the organic phase was dried over anhydrous sodium sulfate, the reaction concentrated, and the residue purified by silica gel column chromatography (dichloromethane/methanol=20/1) to give 2b (500 mg, 79% yield) as a white solid. ESI-MS (m/z): 333.3[ M+H ]] + 。
And a third step of: compound 2b (500 mg,1.50 mmol) was dissolved in dichloromethane (2 mL), and 1, 4-dioxane solution of hydrochloric acid (4M, 1.88 mL) was added dropwise and stirred at room temperature overnight. LCMS monitored the end of the reaction and the reaction concentrated to give 2c as a white solid (300 mg, 74% yield). ESI-MS (m/z): 233.5[ M+H ]] + 。
Fourth step: compound 1i (50 mg, 197umol), compound 2c (68 mg,256 umol) and p-toluenesulfonic acid monohydrate (3.7 mg,19 umol) were dissolved in n-butanol (2 mL) and reacted at microwave 160℃for 2 hours. LCMS monitored the end of the reaction. The reaction solution was purified by reverse phase preparative HPLC to give 2 as a white solid (10.1 mg, yield 11%). ESI-MS (m/z): 449.2[ M+H ] ] + ； 1 H NMR(500MHz,DMSO-d6)δ8.38(d,J＝2.8Hz,1H),7.83(d,J＝8.7Hz,1H),7.46(dd,J＝8.7,2.9Hz,1H),6.91(d,J＝6.9Hz,1H),5.05-5.00(m,1H),4.46-4.42(m,1H),4.12(q,J＝6.9Hz,1H),4.03-3.99(m,2H),3.28-3.25(m,1H),2.95(s,3H),2.50-2.38(m,4H),1.97-1.87(m,1H),1.84-1.77(m,1H),1.23(d,J＝6.7Hz,3H)。
Example 11
(S) -4, 5-dimethyl-2- (((1S, 3 r) -3- (((6- (trifluoromethyl) pyridin-3-yl) oxo) methyl) cyclobutyl) amino) -4,5,9, 10-tetrahydro-6 h,8 h-pyrido [3,2, 1-des ] pteridin-6-one
Compound 11 was prepared by the following steps:
the first step: tert-butyl cis-3-hydroxymethyl cyclobutylcarbamate 11a (150 mg,0.745 mmol), methylsulfonic anhydride (299 mg,1.49 mmol) and N, N-diisopropylethylamine (385 mg,2.98 mmol) were dissolved in dichloromethane (5 mL) and stirred overnight at room temperature. TLC monitored the end of the reaction, the reaction was diluted with dichloromethane, washed successively with water and saturated brine, and the organic phase was dried over anhydrous sodium sulfate, filtered and concentrated to give 11b as a yellow solid (204 mg, yield 98%). 1 H NMR(500MHz,DMSO-d6)δ7.10(d,J＝8.1Hz,1H),4.11(d,J＝5.2Hz,2H),3.19(d,J＝1.6Hz,1H),3.16(d,J＝1.6Hz,3H),2.26(d,J＝5.9Hz,1H),1.68(d,J＝8.7Hz,2H),1.57-1.47(m,2H),1.37(s,9H)。
And a second step of: compound 11b (204 mg,731 mmol), compound 2a (131 mg,0.8 mmol) and cesium carbonate (480 mg,1.49 mmol) were dissolved in N, N-dimethylformamide (5 mL) and stirred overnight at 90 ℃. TLC monitored the end of the reaction, the reaction was diluted with water, extracted with ethyl acetate, washed with saturated brine, the organic phase was dried over anhydrous sodium sulfate, filtered and concentrated, and the residue was purified by silica gel column chromatography (petroleum ether/ethyl acetate=3/1) to give 11c (185 mg, yield 72%) as a yellow solid. ESI-MS (m/z): 347.2[ M+H ] ] + 。
And a third step of: compound 11c (120 mg, 346. Mu. Mol) was dissolved in methylene chloride (5 mL), and a 4M dioxane solution of hydrochloric acid (0.87 mL) was added at 0deg.C and stirred overnight. TLC monitored the end of the reaction and was directly spin-dried to give 11d (85 mg, 99% yield) as a white solid. ESI-MS (m/z): 247.4[ M+H ]] + 。
Fourth step: compound 11d (82 mg, 336. Mu. Mol), compound 1i (85 mg, 336. Mu. Mol) and p-toluenesulfonic acid monohydrate (5.7 mg, 33.6. Mu. Mol) were dissolved in n-butanol (3 mL) and reacted at microwave 160℃for 3 hours. LCMS monitored the end of the reaction. The reaction solution was purified by reverse phase preparative HPLC to give 11 as a white solid (25 mg, 16% yield). ESI-MS (m/z): 463.3[M+H] + ； 1 H NMR(500MHz,DMSO-d6)δ8.47(d,J＝3.0Hz,1H),7.85(d,J＝8.5Hz,1H),7.64(dd,J＝9.0,3.0Hz,1H),6.81(br s,1H),4.32-4.23(m,1H),4.16-4.09(m,3H),4.08-4.01(m,1H),3.30-3.24(m,2H),2.95(s,3H),2.48-2.38(m,3H),1.97-1.88(m,1H),1.86-1.73(m,3H),1.23(d,J＝7.0Hz,3H)。
Example 12
(S) -2- (((1S, 3 r) -3- (((1-cyclopropyl-3- (trifluoromethyl) -1H-pyrazol-5-yl) oxy) methyl) cyclobutyl) amino) -4, 5-dimethyl-4, 5,9, 10-tetrahydro-6H, 8H-pyrido [3,2, 1-des ] pteridin-6-one
Compound 12 was prepared by the following steps:
the first step: ethyl trifluoroacetoacetate 12a (1.7 g,9.21 mmol) and cyclopropylhydrazine hydrochloride 12b (1.0 g,9.21 mmol) were dissolved in 20mL of ethanol and reacted overnight at 80 ℃. The reaction solution was concentrated, and the residue was slurried with petroleum ether and filtered to give compound 12c (800 mg, yield 45%) as a brown solid. ESI-MS (m/z): 193.2[ M+H ] ] + 。
And a second step of: compound 11b (872 mg,3.12 mmol), compound 12c (500 mg,2.61 mmol) and cesium carbonate (1.70 g,5.22 mmol) were dissolved in N, N-dimethylformamide (20 mL) and stirred overnight at 90 ℃. TLC monitored the end of the reaction, the reaction was diluted with water, extracted with ethyl acetate, washed with saturated brine, the organic phase was dried over anhydrous sodium sulfate, filtered and concentrated, and the residue was purified by silica gel column chromatography (petroleum ether/ethyl acetate=3/1) to give 12d (495 mg, yield 52%) as a yellow solid. ESI-MS (m/z): 362.6[ M+H ]] + 。
And a third step of: compound 12d (495mg, 1.1 mmol) was dissolved in dichloromethane (30 mL), and a 4M dioxane solution of hydrochloric acid (1.37 mL) was added at 0deg.C and stirred overnight. TLC monitored the end of the reaction and was directly spin-dried to give 12e as a white solid (231 mg, 80% yield). ESI-MS(m/z):262.6[M+H] + . Fourth step: compound 12e (26 mg,95 umol), compound 1i (20 mg,79 umol) and p-toluenesulfonic acid monohydrate (0.4 mg,7.9 umol) were dissolved in n-butanol (3 mL) and reacted at microwave 160℃for 3 hours. LCMS monitored the end of the reaction. The reaction solution was purified by reverse phase preparative HPLC to give 12 as a white solid (13 mg, yield 35%). ESI-MS (m/z): 492.4[ M+H ]] + ； 1 H NMR(500MHz,DMSO-d6)δ6.67(br s,1H),6.17(s,1H),4.26-4.18(m,1H),4.15-4.06(m,2H),4.05-3.97(m,1H),3.54-3.21(m,5H),2.94(s,3H),2.60-2.50(m,2H),2.47-2.35(m,3H),1.96-1.86(m,1H),1.85-1.70(m,3H),1.21(d,J＝6.8Hz,3H),1.05-0.90(m,4H)。
Example 13
(S) -2- (((1S, 3R) -3- (((1-cyclopropyl-3- (trifluoromethyl) -1H-pyrazol-5-yl) oxy) methyl) cyclobutyl) amino) -5- (hydroxymethyl) -4, 5-dimethyl-4, 5,9, 10-tetrahydro-6H, 8H-pyrido [3,2, 1-des ]
Pteridin-6-oneCompound 13 was prepared by the following steps:
the first step: 2, 4-dichloropyrido [3,2-d ]]Pyrimidine 1f (4.0 g,20.0 mmol) and 2-methyl-L-serine methyl ester hydrochloride 13a (4.07 g,24.0 mmol) were dissolved in dichloromethane (30 mL), N-diisopropylethylamine (7.75 g,59.99mmol,10.45 mL) was added, and stirred overnight at room temperature. LCMS monitored the end of the reaction, the reaction was diluted with dichloromethane, washed with water and saturated brine, respectively, the organic phase was dried over anhydrous sodium sulfate and concentrated by filtration to give 13b as a white solid (5.0 g, 84% yield). ESI-MS (m/z): 297.3[ M+H ]] + 。
And a second step of: compound 13b (5.0 g,16.85 mmol) was dissolved in tetrahydrofuran (50 mL), aqueous hydrochloric acid (6M, 5.62 mL) and platinum dioxide (382 mg,1.69 mmol) were added, the reaction system was replaced with hydrogen gas with a hydrogen balloon, and stirred at room temperature under hydrogen balloon pressure for 48 hours, and LCMS monitored the end of the reaction. The reaction solution was diluted with methanol,filtration and concentration of the filtrate gave 13c (4.0 g, 88% yield) as a white solid. ESI-MS (m/z): 269.3[ M+H ]] + 。
And a third step of: compound 13c (1.5 g,5.58 mmol) and methyl iodide (1.58 g,11.16 mmol) were dissolved in acetonitrile (5 mL), cesium carbonate (3.64 g,11.16 mmol) was added, and the reaction mixture was stirred at room temperature for 48 hours. LCMS monitored the end of the reaction. The reaction solution was diluted with ethyl acetate, each of the solutions was washed with water and saturated brine, and the organic phase was dried over anhydrous sodium sulfate, filtered, and concentrated. The residue was purified by silica gel column chromatography (dichloromethane/methanol=10/1) to give 13d (1.1 g, yield 69%) as a yellow solid. ESI-MS (m/z): 283.3[ M+H ] ] + 。
Fourth step: compound 12e (25 mg,84 umol), compound 13d (20 mg,70.7 umol) and p-toluenesulfonic acid monohydrate (0.4 mg,7.07 umol) were dissolved in n-butanol (3 mL) and reacted at microwave 160℃for 3 hours. LCMS monitored the end of the reaction. The reaction solution was purified by reverse phase preparative HPLC to give 13 as a white solid (13 mg, yield 35%). ESI-MS (m/z): 522.3[ M+H ]] + ； 1 H NMR(500MHz,DMSO-d6)δ6.54(br s,1H),6.17(s,1H),5.06(t,J＝5.5Hz,1H),4.29-4.18(m,1H),4.10(d,J＝4.9Hz,2H),3.76-3.67(m,2H),3.65-3.58(m,1H),3.57-3.49(m,2H),2.97(s,3H),2.55-2.45(m,2H),2.44-2.32(m,3H),1.88-1.72(m,4H),1.33(s,3H),0.98(d,J＝7.8Hz,4H)。
Example 14
(S) -5- (hydroxymethyl) -4, 5-dimethyl-2- (((1S, 3R) -3- (((2-methyl-6- (trifluoromethyl) pyridin-3-yl) oxo) methyl) cyclobutyl) amino) -4,5,9, 10-tetrahydro-6H, 8H-pyrido [3,2, 1-des-tins]Pteridin-6-oneCompound 14 was prepared by the following steps:
the first step: compound 2a (2.0 g,12.26 mmol), na 2 CO 3 (2.6 g,24.52 mmol) was added to water (60 mL), dissolved by stirring, elemental iodine (3.11 g,12.26 mmol) was added to the reaction solution, and stirred at room temperatureMix for 3 hours. LCMS monitored the disappearance of starting material, pH of the reaction solution was adjusted to 5-6 with dilute hydrochloric acid and extracted with ethyl acetate. The organic phases were combined, dried over anhydrous sodium sulfate, concentrated by filtration, and the residue was chromatographed on silica gel (ethyl acetate/petroleum ether=0-30% gradient elution) to give 14a as a white solid (1.5 g, 42% yield). ESI-MS (m/z): 290.2[ M+H ]] + 。
And a second step of: compound 14a (0.5 g,1.73 mmol) was dissolved in DMF (5 mL) and K was added 2 CO 3 (264 mg,2.64 mmol), stirred at 50℃for 2 hours, LCMS monitored translation for completion. The reaction solution was diluted with water, extracted with ethyl acetate, and the organic phases were combined, dried over anhydrous sodium sulfate, filtered and concentrated. Column chromatography of the residue on silica gel (ethyl acetate/petroleum ether=0-15% gradient elution) gives 14b as a white solid (0.5 g, 76% yield). ESI-MS (m/z): 380.2[ M+H ]] + 。
And a third step of: compound 14b (412 mg,1.09 mmol), palladium acetate (24 mg, 109. Mu. Mol), trimethylcyclotriboroxine (682 mg,5.43 mmol), tricyclohexylphosphine (152 mg, 803. Mu. Mol), tripotassium phosphate (922 mg,4.35 mmol), water (3 mL), 1, 4-dioxane (30 mL) were added to a 100mL two-necked flask, after nitrogen substitution, stirring was carried out at 90℃overnight, TLC was monitored for the end of the reaction, the reaction solution was diluted with water, filtered with celite, extracted with ethyl acetate, and finally washed with saturated brine, the organic phase was dried over anhydrous sodium sulfate, filtered and concentrated, and the residue was purified by silica gel column chromatography (petroleum ether/ethyl acetate=10/1) to give yellow liquid 14c (198 mg, yield 68%). ESI-MS (m/z): 268.2[ M+H ] +.
Fourth step: compound 14c (198mg, 741 umol) was dissolved in dichloromethane (10 mL), boron tribromide (928 mg,3.71 umol) was added dropwise at-78 ℃ and stirred for 1 hour, TLC monitored the end of the reaction, the reaction solution was diluted with water, extracted with dichloromethane, the organic phase was dried over anhydrous sodium sulfate, filtered and concentrated, and the residue was purified by silica gel column chromatography (petroleum ether/ethyl acetate=1/1) to give yellow liquid 14d (143 mg, yield 91%). ESI-MS (m/z): 178.4[ M+H ] +.
Fifth step: compound 14d (120 mg,677 umol), compound 11b (208 mg,745 umol) and cesium carbonate (441 mg,1.35 mmol) were dissolved in N, N-dimethylformamide (5 mL),stirred overnight at 90 ℃. TLC monitored the end of the reaction, the reaction was diluted with water, extracted with ethyl acetate, washed with saturated brine, the organic phase was dried over anhydrous sodium sulfate, filtered and concentrated, and the residue was purified by silica gel column chromatography (petroleum ether/ethyl acetate=3/1) to give 14e (196 mg, yield 80%) as a yellow solid. ESI-MS (m/z): 361.4[ M+H ]] + 。
Sixth step: compound 14e (196 mg, 543. Mu. Mol) was dissolved in dichloromethane (10 mL), 4M hydrochloric acid (0.952 mL) was added at 0deg.C and stirred overnight. TLC monitored the end of the reaction and was directly spin-dried to give 14f as a white solid (112 mg, 79% yield). ESI-MS (m/z): 261.3[ M+H ]] + 。
Seventh step: compound 14f (27 mg,106 umol), compound 13d (20 mg,70.7 umol) and p-toluenesulfonic acid monohydrate (0.4 mg,7.07 umol) were dissolved in n-butanol (3 mL) and reacted at microwave 160℃for 3 hours. LCMS monitored the end of the reaction. The reaction solution was purified by reverse phase preparative HPLC to give 14 as a white solid (19 mg, yield 52%). ESI-MS (m/z): 507.3[ M+H ]] + ； 1 H NMR(500MHz,DMSO-d6)δ7.67(d,J＝8.5Hz,1H),7.48(d,J＝8.5Hz,1H),6.54(br s,1H),5.06(t,J＝5.5Hz,1H),4.29-4.19(m,1H),4.05(d,J＝4.8Hz,2H),3.77-3.66(m,2H),3.65-3.60(m,1H),3.58-3.53(m,1H),2.98(s,3H),2.48-2.35(m,8H),1.90-1.74(m,4H),1.33(s,3H)。
Example 25
(S) -4, 5-dimethyl-2- (((1 r, 3S) -3- ((2-methyl-6- (trifluoromethyl) pyridin-3-yl) oxo) cyclobutyl) amino) -4,5,9, 10-tetrahydro-6H, 8H-pyrido [3,2, 1-des ] pteridin-6-one
The intermediate 14d was used in place of the first step 2a of example 2, and in a similar manner and reaction procedure, compound 25 was obtained. The method comprises the following steps:
the first step: compound 1b, compound 14d and cesium carbonate were dissolved in N, N-dimethylformamide and stirred overnight at 80 ℃. LCMS monitored the end of the reaction, the reaction was diluted with ethyl acetate, washed sequentially with water and saturated brine, the organic phase was dried over anhydrous sodium sulfate, the reaction concentrated, and the residue purified by silica gel column chromatography (dichloromethane/methanol=20/1) to give 25b as a white solid.
And a second step of: compound 25b was dissolved in dichloromethane, 1, 4-dioxane hydrochloride solution was added dropwise, and stirred at room temperature overnight. LCMS monitored the end of the reaction and the reaction concentrated to give 25c as a white solid.
And a third step of: compound 1i, compound 25c and p-toluenesulfonic acid monohydrate were dissolved in n-butanol and reacted at microwave 160 ℃ for 2 hours. LCMS monitored the end of the reaction. The reaction was purified by reverse phase prep HPLC to give white solid 25.ESI-MS (m/z): 463.5[ M+H ]] + ； 1 H NMR(500MHz,DMSO-d6)δ7.66(d,J＝8.5Hz,1H),7.25(d,J＝8.5Hz,1H),6.88(d,J＝6.8Hz,1H),4.97(br s,1H),4.50-4.36(m,1H),4.11(q,J＝6.8Hz,1H),4.04-3.98(m,1H),3.35-3.29(m,2H),2.94(s,3H),2.60-2.30(m,9H),2.00-1.88(m,1H),1.85-1.75(m,1H),1.23(d,J＝6.6Hz,3H)。
Biological screening and results of Wnt pathway inhibitors
Test example 1: construction of Colo205-LUC-TCF/LEF-M1 reporter cell line
Colo205 cell line (Proc. Natl. Acad. Sci. Cell bank, cat#TCHu102) was purchased from Proc. Natl. Acad. Sci. Cell bank, amplified and subcultured, and then transfected with a luciferase reporter plasmid (Promega) driven by TCF/LEF transcription factor by lipo3000 liposome transfection in the exponential growth phase of cells. The plasmid carries a resistance gene, and can be used for resistance screening. Transfection was performed in 10cm dishes using conventional complete medium without resistance. After 2 days, the medium with resistance was changed and the culture was continued. The resistant medium was then changed every 2 days and the suspension cells were discarded, and the original medium was centrifuged to remove cells and debris and was then retained as an adaptive medium. After the cells had grown up the dishes, the cells were digested, counted and passaged into 96-well plates to average the number of cells contained in each well to 1.5 cells/well, using adaptation medium at the time of passaging. The remaining cells were frozen. After 4 hours of culture after passage, the cells were allowed to adhere, and then the cell numbers of each well were observed under a microscope. Only 1 cell per well was labeled, which was a monoclonal well. Then, the culture medium was changed every 2 days for normal culture, and observation was performed. The pre-monoclonal cells have wells that continue to grow, and are labeled 2 times, and can be replaced with normal medium with resistance. When cells in a monoclonal well grow over a 96-well plate well, they are digested and passaged to a 24-well culture plate, after the 24-well plate grows over, passaged to 1 96-well plate and 1 6-well plate, wherein 96-well plate cells are passaged to at least 6-well, wherein 3-well are added with a known Wnt inhibitor, and the other 3-well are not treated. After 24 hours, the 96-well plate cells were added with a fluorescent detection reagent to detect the fluorescence intensity. Cell lines in which fluorescence expression was not present in the treatment and post-fluorescence reduction was inhibited were selected and further cultured. The Colo205-LUC-TCF/LEF-M1 cell line is one of the cell lines screened by the screening method, the growth curve, the cell morphology and the cell growth state of the cell line are similar to those of original Colo205 cells, the ratio of the fluorescent signals treated by adding the inhibitor to the fluorescent signals not treated by the inhibitor is large in all the cell lines, and the ratio can be inhibited by 4-5 times at 4h, so that the cell line is completely suitable for screening Wnt inhibitors in the later period.
Test example 2: detection of Colo205-LUC-TCF/LEF M1 reporter cell line inhibition Capacity by Compounds
The Colo205-LUC-TCF/LEF M1 cell strain is a report tool cell for stably transfecting pGL4.49-LUC2-TCF/LEF vectors, the beta-catenin Wnt channel is continuously activated, after an inhibitor is added, the Wnt channel is inhibited, the expression level of firefly luciferase regulated by TCF/LEF cis elements on the vectors is reduced, and after a detection substrate is added subsequently, the detected optical signal is correspondingly reduced, so that the inhibition effect of the compound is detected.
100. Mu.L of compound was added to each well of a 96-well cell culture plate at a maximum concentration of 20. Mu.M, and the compound concentration was diluted 3-fold in a gradient. 10000 stably reporter-transfected colo205 cells and 100. Mu.L medium were then inoculated into each well, with corresponding treatments as positive and negative control wells. Cells were placed in 5% CO 2 The cell incubator is used for culturing for 4 hours at 37 ℃ and removing the culture solution after 4 hoursTo each well, 100. Mu.L of a reagent (Promega) containing a corresponding firefly luciferase substrate was added, and the activity of a luciferase reporter gene was measured. The luminescence intensity was read in full wavelength mode with SpectraMax. The IC of the compound was calculated from the light signal intensity of DMSO-treated cells alone as positive control and the light signal intensity of cell-free wells as negative control 50 Is a concentration of (3). The Colo205 reporter assay data are summarized in Table 1 below.
IC of the Table 1 Compounds against Colo205-LUC-TCF/LEF reporter inhibition 50 Value of
Test example 3: proliferation inhibition assay of compounds on Wnt mutant cell lines (Colo 205, DU4475, NCI-H929 and HepG 2) and non-Wnt mutant cell lines (Hela and RKO)
The cell lines used in the experiments were those in which the Wnt pathway was continuously activated and which proliferated as Wnt pathway dependent Colo205, DU4475, NCI-H929 and HepG2 cell lines; whereas normally Wnt pathway is not activated and HELA and RKO cell lines whose proliferation is independent of Wnt pathway are used as control cell lines, it was judged that the inhibition of Wnt dependent proliferation by the compounds of the invention is not due to other non-specific toxicities.
Colo205, du4475, NCI-H929, hepG2, HELA and RKO cell lines cultured in the respective media were treated in the logarithmic phase, and after collecting the cells, a uniform cell suspension of known concentration was prepared, and then the cell suspension was added to a 96-well cell culture plate so that 1000 cells were contained in each well. Placing 5% CO 2 The cells are cultured in a cell incubator at 37 ℃ for 20-24 hours. The following day, the 3-fold gradient of the compound which had been completely dissolved was added to each cell culture well to give a final maximum concentration of 20. Mu.M in the cell culture well, and the culture was continued for 96 hours. The assay was performed using a Promega cell viability assay, the more cell proliferation the stronger the final signal intensity. The detection instrument is in SpectraMax, full wavelength mode. Wells with DMSO alone served as positive control wells and wells without cells inoculated served as negative control wells, compound vs W was calculated IC for proliferation inhibition of nt sustained activation or proliferation dependent cells 50 Value, and IC for proliferation inhibition of Wnt-unactivated or proliferation-independent cells 50 The inhibition of Wnt pathway and the toxic effect on normal cells were evaluated. The results are shown in Table 2 below.
IC of the compounds of Table 2 for inhibition of proliferation of Wnt mutant cell lines 50 Value of
The above results indicate that the compounds of the present invention have significant inhibitory activity against mutant cell lines Colo205, DU4475, NCI-H929 and HepG2, while having substantially no significant inhibitory activity against Hela and RKO cell lines, indicating that the compounds of the present invention have significant and selective inhibition of the Wnt pathway.

Claims (4)

1. A compound having the structure of formula 25 or a pharmaceutically acceptable salt, isotopic derivative, stereoisomer thereof:
2. a process for the preparation of a compound having the structure of formula 25 as claimed in claim 1 comprising the steps of:
the first step: compound 1b, compound 14d and cesium carbonate were dissolved in N, N-dimethylformamide, stirred at 80 ℃ until the reaction was completed, the reaction solution was diluted with ethyl acetate, washed with water and saturated brine in this order, the organic phase was dried over anhydrous sodium sulfate, the reaction solution was concentrated, and the residue was purified by silica gel column chromatography (dichloromethane/methanol=20/1) to give white solid 25b;
And a second step of: compound 25b is dissolved in dichloromethane, 1, 4-dioxane solution of hydrochloric acid is added dropwise, the mixture is stirred at room temperature until the reaction is finished, and the reaction solution is concentrated to obtain white solid 25c;
and a third step of: compound 1i, compound 25c and p-toluenesulfonic acid monohydrate were dissolved in n-butanol and reacted at 160 ℃ under microwave to completion, and the reaction solution was purified by reverse phase preparative HPLC to give white solid 25.
3. A pharmaceutical composition comprising a compound of claim 1 or a pharmaceutically acceptable salt, isotopic derivative, stereoisomer thereof.
4. Use of a compound according to claim 1 or a pharmaceutically acceptable salt, isotopic derivative, stereoisomer thereof, or a pharmaceutical composition according to claim 3 for the manufacture of a medicament for the prevention and/or treatment of cancer, tumor, inflammatory disease, autoimmune disease or immune-mediated disease.
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